Expressed Sequence Tags for Bovine Muscle Satellite Cells,Myotube Formed-Cells and Adipocyte-Like Cells

Background

Muscle satellite cells (MSCs) represent a devoted stem cell population that is responsible for postnatal muscle growth and skeletal muscle regeneration. An important characteristic of MSCs is that they encompass multi potential mesenchymal stem cell activity and are able to differentiate into myocytes and adipocytes. To achieve a global view of the genes differentially expressed in MSCs, myotube formed-cells (MFCs) and adipocyte-like cells (ALCs), we performed large-scale EST sequencing of normalized cDNA libraries developed from bovine MSCs.

Results

A total of 24,192 clones were assembled into 3,333 clusters, 5,517 singletons and 3,842contigs. Functional annotation of these unigenes revealed that a large portion of the differentially expressed genes are involved in cellular and signaling processes. Database for Annotation, Visualization and Integrated Discovery (DAVID) functional analysis of three subsets of highly expressed gene lists (MSC233, MFC258, and ALC248) highlighted some common and unique biological processes among MSC, MFC and ALC. Additionally, genes that may be specific to MSC, MFC and ALC are reported here, and the role of dimethylarginine dimethylaminohydrolase2 (DDAH2) during myogenesis and hemoglobin subunit alpha2 (HBA2) during transdifferentiation in C2C12 were assayed as a case study. DDAH2 was up-regulated during myognesis and knockdown of DDAH2 by siRNA significantly decreased myogenin (MYOG) expression corresponding with the slight change in cell morphology. In contrast, HBA2 was up-regulated during ALC formation and resulted in decreased intracellular lipid accumulation and CD36 mRNA expression upon knockdown assay.

Conclusion

In this study, a large number of EST sequences were generated from the MSC, MFC and ALC. Overall, the collection of ESTs generated in this study provides a starting point for the identification of novel genes involved in MFC and ALC formation, which in turn offers a fundamental resource to enable better understanding of the mechanism of muscle differentiation and transdifferentiation.     

The N-end rule proteolytic system in autophagy

The N-end rule pathway is a cellular proteolytic system that utilizes specific N-terminal residues as degradation determinants, called N-degrons. N-degrons are recognized and bound by specific recognition components (N-recognins) that mediate polyubiquitination of low-abundance regulators and selective proteolysis through the proteasome. Our earlier work identified UBR4/p600 as one of the N-recognins that promotes N-degron-dependent proteasomal degradation. In this study, we show that UBR4 is associated with cellular cargoes destined to autophagic vacuoles and is degraded by the lysosome. UBR4 loss causes multiple misregulations in autophagic pathways, including an increased formation of LC3 puncta. UBR4-deficient mice die during embryogenesis primarily due to defective vascular development in the yolk sac (YS), wherein UBR4 is associated with a bulk lysosomal degradation system that absorbs maternal proteins from the YS cavity and digests them into amino acids. Our results suggest that UBR4 plays a role not only in selective proteolysis of short-lived regulators through the proteasome, but also bulk degradation through the lysosome. Here, we discuss a possible mechanism of UBR4 as a regulatory component in the delivery of cargoes destined to interact with the autophagic core machinery.     

Effects of Ethanol Exposure During Early Pregnancy in Hyperactive, Inattentive and Impulsive Behaviors and MeCP2 Expression in Rodent Offspring

Prenatal exposure to alcohol has consistently been associated with adverse effects on neurodevelopment, which is collectively called fetal alcohol spectrum disorder (FASD). Increasing evidence suggest that prenatal exposure to alcohol increases the risk of developing attention deficit/hyperactivity disorder-like behavior in human. In this study, we investigated the behavioral effects of prenatal exposure to EtOH in offspring mice and rats focusing on hyperactivity and impulsivity. We also examined changes in dopamine transporter and MeCP2 expression, which may underlie as a key neurobiological and epigenetic determinant in FASD and hyperactive, inattentive and impulsive behaviors. Mouse or rat offspring born from dam exposed to alcohol during pregnancy (EtOH group) showed hyper locomotive activity, attention deficit and impulsivity. EtOH group also showed increased dopamine transporter and norepinephrine transporter level compared to control group in the prefrontal cortex and striatum. Prenatal exposure to EtOH also significantly decreased the expression of MeCP2 in both prefrontal cortex and striatum. These results suggest that prenatal exposure to EtOH induces hyperactive, inattentive and impulsive behaviors in rodent offspring that might be related to global epigenetic changes as well as aberration in catecholamine neurotransmitter transporter system.     

The structure of a shellfish specific GST class glutathione S‐transferase from antarctic bivalve Laternula elliptica reveals novel active site architecture

Glutathione‐S‐transferases have been identified in all the living species examined so far, yet little is known about their function in marine organisms. In a previous report, the recently identified GST from Antarctic bivalve Laternula elliptica (LeGST) was classified into the rho class GST, but there are several unique features of LeGST that may justify reclassification, which could represent specific shellfish GSTs. Here, we determined the crystal structure of LeGST, which is a shellfish specific class of GST. The structural analysis showed that the relatively open and wide hydrophobic H‐site of the LeGST allows this GST to accommodate various substrates. These results suggest that the H‐site of LeGST may be the result of adaptation to their environments as sedentary organisms. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Rhamnetin and Cirsiliol Induce Radiosensitization and Inhibition of Epithelial-Mesenchymal Transition (EMT) by miR-34a-mediated Suppression of Notch-1 Expression in Non-small Cell Lung Cancer Cell Lines

Radioresistance is a major cause of decreasing the efficiency of radiotherapy for non-small cell lung cancer (NSCLC). To understand the radioresistance mechanisms in NSCLC, we focused on the radiation-induced Notch-1 signaling pathway involved in critical cell fate decisions by modulating cell proliferation. In this study, we investigated the use of Notch-1-regulating flavonoid compounds as novel therapeutic drugs to regulate radiosensitivity in NSCLC cells, NCI-H1299 and NCI-H460, with different levels of radioresistance. Rhamnetin and cirsiliol were selected as candidate Notch-1-regulating radiosensitizers based on the results of assay screening for activity and pharmacological properties. Treatment with rhamnetin or cirsiliol reduced the proliferation of NSCLC cells through the suppression of radiation-induced Notch-1 expression. Indeed, rhamnetin and cirsiliol increased the expression of tumor-suppressive microRNA, miR-34a, in a p53-dependent manner, leading to inhibition of Notch-1 expression. Consequently, reduced Notch-1 expression promoted apoptosis through significant down-regulation of the nuclear factor-κB pathway, resulting in a radiosensitizing effect on NSCLC cells. Irradiation-induced epithelial-mesenchymal transition was also notably attenuated in the presence of rhamnetin and cirsiliol. Moreover, an in vivo xenograft mouse model confirmed the radiosensitizing and epithelial-mesenchymal transition inhibition effects of rhamnetin and cirsiliol we observed in vitro. In these mice, tumor volume was significantly reduced by combinational treatment with irradiation and rhamnetin or cirsiliol compared with irradiation alone. Taken together, our findings provided evidence that rhamnetin and cirsiliol can act as promising radiosensitizers that enhance the radiotherapeutic efficacy by inhibiting radiation-induced Notch-1 signaling associated with radioresistance possibly via miR-34a-mediated pathways.